Induction, propagation and isolation of liver progenitor cells

ABSTRACT

The present invention relates to methods of induction and isolation of progenitor cells from stem cell cultures, specifically liver progenitor cells from human embryonic stem cell cultures. In one embodiment, the present invention provides a method of inducing hepatocyte-like progenitor cells by placing a quantity of human embryonic stem cells in a medium supplemented with an inhibitor of the MAPK/MEK/ERK signaling pathway, FGFR, GSK3 and/or BMP.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/556,478, filed on Sep. 9, 2009. The present application claims the benefit of priority under 35 U.S.C. §119(e) of provisional application, U.S. App. No. 61/095,494, filed Sep. 9, 2008, the contents of which are hereby incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with U.S. Government support on behalf of the NIH Pilot Project Grant RCLD. The U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to the field of stem cells and, more specifically, to the induction, propagation and isolation of progenitor cells of the liver.

BACKGROUND

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

A stem cell is a cell type that has a unique capacity to renew itself and give rise to specialized or differentiated cells. Although most cells of the body are committed to conduct a specific function, a stem cell is uncommitted, until it receives a signal to develop into a specialized cell type. What makes the stem cells unique is their proliferative capacity, combined with their ability to become specialized. Somatic stem cells are present in the adult organism. Pluripotency tests have shown that whereas the embryonic or blastocyst-derived stem cells can give rise to all cells in the organism, including the germ cells, somatic stem cells have a more limited repertoire in descendent cell types.

To date, some studies have shown that human embryonic stem (“ES”) cells can be induced to differentiate into cells with gene expression profiles characteristic of hepatocytes on a modest scale. However, there remains a need in the art for novel methods of actually inducing liver stem or progenitor cells from human ES cell culture. Large scale production of hepatocyte precursors from human ES cells has application in several key areas; for example, to enable in vitro studies of the pathogenesis of liver disease, such as viral hepatitis; to enable studies of the metabolism and toxicity of drugs in vitro, leading to better predictive toxicology tests; and to provide cells for transplantation therapy in liver diseases such as Hepatitis C, which is currently a worldwide epidemic affecting over 200 million patients.

Thus, there is a need in the art for novel methods of inducing liver progenitor cells from stem cells.

SUMMARY OF THE INVENTION

Various embodiments include a method of inducing and/or propagating a quantity of progenitor cells, including providing a quantity of human stem cells, and placing the quantity of human stem cells in a medium supplemented with one or more of an inhibitor of the mitogen-activated protein kinase (MAPK)/map-erk kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, a fibroblast growth factor receptor (FGFR) inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor. In another embodiment, the human stem cells are human embryonic stem cells. In another embodiment, the inhibitor of the MAPK/MEK/ERK signaling pathway includes a compound of the formula:

or a pharmaceutical equivalent, analog, derivative and/or salt thereof. In another embodiment, the inhibitor of the MAPK/MEK/ERK signaling pathway includes a compound of the formula:

or a pharmaceutical equivalent, analog, derivative and/or salt thereof. In another embodiment, the FGFR inhibitor includes a compound of the formula:

or a pharmaceutical equivalent, analog, derivative and/or salt thereof. In another embodiment, the GSK3 inhibitor includes a compound of the formula:

or a pharmaceutical equivalent, analog, derivative and/or salt thereof.

In another embodiment, the progenitor cells are hepatocyte-like. In another embodiment, the stem cell includes human embryonic stem cell 2 (HES2) and/or human embryonic stem cell 3 (HES3). In another embodiment, the medium includes 0.2 to 3.0 μM of the inhibitor of the MAPK/MEK/ERK signaling pathway. In another embodiment, the medium includes 1.0 to 6.0 μM of the FGFR inhibitor. In another embodiment, the medium includes 1.0 to 6.0 μM of the GSK3 inhibitor. In another embodiment, the medium is further including a bone morphogenetic protein (BMP) inhibitor. In another embodiment, the medium includes 1.0 to 6.0 μM of the BMP inhibitor. In another embodiment, the BMP inhibitor includes dorsomorphin.

Other embodiments include a composition including an inhibitor of the MAPK/MEK/ERK signaling pathway, an FGFR inhibitor, a GSK3 inhibitor, and a BMP inhibitor.

Various embodiments also include a composition including activin, BMP, epidermal growth factor (EGF), heregulin, fibroblast growth factor 1 (FGF-1), insulin growth factor (IGF), vascular endothelial growth factor (VEGF), angiopoietin, and transforming growth factor beta (TGF beta).

Other embodiments include a composition, including a quantity of progenitor cells, produced by the process providing a quantity of human stem cells, and placing the quantity of human stem cells in a medium supplemented with one or more of an inhibitor of the mitogen-activated protein kinase (MAPK)/map-erk kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, a fibroblast growth factor receptor (FGFR) inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor.

Other embodiments include a method of inducing and/or propagating a quantity of endoderm progenitor cells, including providing a quantity of human pluripotent stem cells, placing the quantity of human pluripotent stem cells in a medium supplemented with an inhibitor of MKK1/2, a fibroblast growth factor receptor (FGFR) inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor, and culturing the human pluripotent stem cells to form endoderm progenitor cells. In one embodiment, the human pluripotent stem cells are human embryonic stem cells. In another embodiment, the inhibitor of MKK1/2 includes a compound of the formula:

or a salt thereof. In another embodiment, the inhibitor of MKK1/2 includes a compound of the formula:

or a salt thereof. In another embodiment, the FGFR inhibitor includes a compound of the formula:

or a salt thereof. In another embodiment, the GSK3 inhibitor includes a compound of the formula:

or a salt thereof.

In another embodiment, the endoderm progenitor cells are hepatocyte-like. In another embodiment, the human pluripotent stem cells includes human embryonic stem cell 2 (HES2) and/or human embryonic stem cell 3 (HES3). In another embodiment, the medium includes 0.2 to 3.0 μM of the inhibitor of MKK1/2. In another embodiment, the medium includes 1.0 to 6.0 μM of the FGFR inhibitor. In another embodiment, the medium includes 1.0 to 6.0 μM of the GSK3 inhibitor. In another embodiment, the medium further includes a bone morphogenetic protein (BMP) inhibitor. In another embodiment, the medium includes 1.0 to 6.0 μM of the BMP inhibitor. In another embodiment, the inhibitor includes dorsomorphin.

Other embodiments include a composition including an inhibitor of MKK1/2, an FGFR inhibitor, a GSK3 inhibitor, and a BMP inhibitor. In another embodiment, the composition including activin, BMP, epidermal growth factor (EGF), heregulin, fibroblast growth factor 1 (FGF-1), insulin growth factor (IGF), vascular endothelial growth factor (VEGF), angiopoietin, and transforming growth factor beta (TGF beta). In another embodiment, the composition, including a quantity of endoderm progenitor cells, produced by the process of claim 1.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts, in accordance with an embodiment herein, charts of gene expression in induced cells. FIG. 1 shows that the large cells do not express pluripotency markers, but express markers of definitive endoderm (Sox-17), extraembryonic endoderm (Sox-7, Gata-4, AFP), and gut derivatives (Pax-6, pancreas; Cdx-2, intestine; albumin, liver; FoxA2 and HNF-6, liver progenitors).

FIG. 2 depicts, in accordance with an embodiment herein, 3i media induced expression of (a) albumin and HNF1b and (b) HNF-6.

FIG. 3 depicts, in accordance with an embodiment herein, immunostaining for endoderm and liver markers.

FIG. 4 depicts, in accordance with an embodiment herein, isolation and attachment of GCTM-5 positive cells from adult liver, including (a) Day 4 in Kubota's media+bFGF 5 ng/ml, and (b) Day 13 in Kubota's media+bFGF 5 ng/ml.

DESCRIPTION OF INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., J. Wiley & Sons (New York, N.Y. 1992); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

“Stem cell” as used herein refers to a cell that can continuously produce unaltered daughters and also has the ability to produce daughter cells that have different, more restricted properties. Stem cells include adult and ES cells.

“Progenitor cell” as used herein refers to a parent cell that gives rise to a distinct cell lineage by a series of cell divisions.

“Hepatocyte-like” cells as used herein includes liver cells, as well as cells that have attributes and characteristics of liver cells, such as the expression of markers associated with liver progenitor cells.

“HES2” and “HES3” as used herein refers to cell lines of human embryonic stem cells.

“Packaging material” as used herein refers to one or more physical structures used to house the contents of a kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment.

“Package” as used herein refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding individual kit components. Thus, for example, a package can be a cryocontainer used to contain suitable quantities of peritoneal stem cells and/or peritoneal cells described herein. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.

As used herein, “FGF” means fibroblast growth factor.

As used herein, “MEK” (map-erk kinase), “MAPK” (mitogen-activated protein kinase) and “ERK” (extracellular signal-regulated kinase) refers to mitogen activated kinases.

As used herein, the term “MAPK/MEK/ERK signaling pathway” refers to a signal transduction pathway involving MAPK, MEK and ERK mitogen activated kinases, coupling intracellular responses to the binding of growth factors to cell surface receptors. The term MAPK/MEK/ERK signaling pathway includes the many protein components and kinase cascades that are part of the signaling pathway, as well as the various targets regulated by the pathway.

As used herein, the term “GSK” means glycogen synthase kinase 3 (GSK3).

As used herein, the term “BMP” means bone morphogenetic protein.

As readily apparent to one of skill in the art, any number of commercially and non commercially available molecules and compounds may be used to inhibit various signaling pathways and components referenced herein. For example, as used herein, FGF inhibitors include PD173074, PLX052, PKC412, AZD2171, Ki23057, BIBF1120, CHIR-258, Mastinib (AB1010), PHA739358, BMS-582664, AZD2171, PRO-001, Tyrphostin A46, and/or Tyrphostin B40. Similarly, MAPK/MEK/ERK signaling pathway inhibitors include PD98059, U0126, 5-Indotubercidin,

terna Zentaris, PD98059, AG 99, Apigenin, SP600125, 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, SL327, SU4984, FR180204, SB203580, PD169316, SB202190, and/or ERK activation inhibitor peptides (such as Ste-PKKKPTPIQLNP-NH₂). Inhibitors of the GSK3 include BIO and related products, CT99021, Aloisine, RP106, Aloisine A, TDZD-8, OTDZT, AR-A014418, 7AIPM, Neurogenesis Inducer, TWS119, Kenpaullone (NSC-664704), Indirubin-3′-monoxime, GSK3b inhibitor peptide (such as Myr-N-GKEAPPAPPQSpP-NH₂), and/or Wnt signaling ligands (such as Wnt1 and/or Wnt3a). Inhibitors of BMP include LDN-193189, and/or BMP inhibitor proteins (such as Noggin, Chordin, Follistatin, and/or Wif).

As used herein, the term “PD184352” means Formula 1, a selective and non-competitive inhibitor of map-erk kinase 1.

As used herein, the term “PD0325901” means Formula 2, a specific non-ATP-competitive inhibitor of map-erk kinase.

As used herein, the term “SU5402”, or 3-[3-(2-Carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone, means Formula 3, an inhibitor of the tyrosine kinase activity of fibroblast growth factor receptor 1.

As used herein, the term “CHIR99021” means Formula 4, a selective inhibitor of GSK3β.

As disclosed herein, the inventors developed a method for the induction, isolation, and propagation of liver progenitor cells from human ES cell cultures or other human pluripotent stem cells, including induced pluripotent stem cells or pluripotent stem cells derived from fetal or adult tissues including germ cells. Human ES cell cultures, maintained as described previously in serum containing medium in the presence of fibroblast feeder cell support, were transferred to defined medium supplemented with small molecule inhibitors of MEK kinase, GSK-3, and FGF signaling. This combination of inhibitors has been shown to maintain pluripotency in cultures of mouse ES cells, but paradoxically, when added to cultures of human ES cells, it induces differentiation and the appearance of cells with morphology of primitive hepatocyte precursors. These cultures contain cells that are positive for the GCTM-5 antigen and may coexpress EpCAM and NCAM along with GCTM-5 on their surface. This combination of antigens enables the isolation of pure populations of progenitors from the differentiating cultures by flow cytometry or magnetic bead purification. These cells may also co-express markers of the bile duct lineage, such as cytokeratin 19, or markers of hepatocytes, such as amylase, albumin, or other liver specific gene products. The cells propagate and undergo expansion for prolonged periods in the presence of this medium described below, or in Kubota's medium, a minimal formulation previously shown to promote growth of adult liver progenitor cells in vitro. The cells are positive for surface markers characteristic of liver progenitor cells in the adult including the GCTM-5 antigen, Ep-CAM, and N-CAM. They are capable of differentiation into cells expressing markers of either bile duct or hepatocytes, such markers including albumin, transthyretin, cytochrome P450 enzymes. They show functional activity of adult liver cells such as indocyanine green dye uptake. They can give rise to mature hepatocytes either in vitro or following transplantation into liver damage models in vivo.

In one embodiment, the present invention provides a method of inducing and/or propagating liver progenitor cells by placing an ES cell culture in a medium supplemented with small molecule inhibitors of MEK kinase, GSK-3 and/or FGF signaling. In another embodiment, the ES cells are from a human. In another embodiment, the ES cell culture includes HES2 and/or HES3 cells.

In one embodiment, the present invention provides a method of inducing and/or propagating hepatocyte precursors by placing pluripotent stem cells in a medium supplemented with small molecule inhibitors of MEK kinase, GSK-3 and/or FGF signaling. In another embodiment, the pluripotent stem cells are induced pluripotent stem cells. In another embodiment, the pluripotent stem cells are derived from fetal and/or adult tissues. In another embodiment, the pluripotent stem cells are derived from germ cells. In another embodiment, the hepatocyte precursors may be used for in vitro studies of the pathogenesis of liver disease, including viral hepatitis. In another embodiment, the hepatocyte precursors are used for studies of metabolism and toxicity of drugs in vitro, including the development of predictive toxicology tests. In another embodiment, the hepatocyte precursors provide cells for transplantation therapy in liver diseases, including Hepatitis C. In another embodiment, the hepatocyte precursors express GCTM-5, EpCAM and/or NCAM. In another embodiment, the hepatocyte precursors express markers of the bile duct lineage. In another embodiment, markers of the bile duct lineage include cytokeratin 19. In another embodiment, the hepatocyte precursors express liver specific gene products, including amylase and/or albumin.

In one embodiment, the present invention provides a method of isolating progenitor cells by detecting the expression of GCTM-5, EpCAM and/or NCAM. In another embodiment, expression of GCTM-5, EpCAM and/or NCAM is detected by flow cytometry and/or magnetic bead purification.

In one embodiment, the present invention provides a method of isolating pure populations of progenitors from differentiating cultures by detecting the expression of GCTM-5, EpCAM, NCAM, markers of the bile duct lineage, and/or liver specific gene products. In another embodiment, expression is detected by flow cytometry and/or magnetic bead purification.

In one embodiment, the present invention provides a method of propagating adult liver cells by maintaining a stem cell culture in a 3i and/or Kubota medium. In another embodiment, the adult liver cells have the capacity of indocyanine green dye uptake.

In one embodiment, the present invention provides a culture medium. In one embodiment, the culture medium is 3i medium, including Neuro basal medium about 30 to 60%, DMEM/F-12 about 30-60%, N2 supplement about 1/100, 1/200, 1/300, 1/400, or 1/500 v/v, B27 supplement about 1/50, 1/100, 1/200, 1/300, 1/400, or 1/500 v/v, 100 mM L-glutamine about 1/50, 1/100, 1/200, 1/300, 1/400, or 1/500 v/v, 0.1M β-ME about 1/500, 1/1000, 1/1500, or 1/2000 v/v, SU5402 (FGFR inhibitor) about 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, PD184352 (ERK cascade inhibitor) about 0.5 μM, 0.8 μM, 1 μM, 2 μM, CHIR99021 (GSK3 inhibitor) about 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM. In another embodiment, the culture medium is 3i medium, including Neuro basal medium 50%, DMEM/F-12 50%, N2 supplement 1/200 v/v, B27 supplement 1/100 v/v, 100 mM L-glutamine 1/100 v/v, 0.1 M β-ME 1/1000 v/v, SU5402 (FGFR inhibitor) 2 μM, PD184352 (ERK cascade inhibitor) 0.8 μM, CHIR99021 (GSK3 inhibitor) 3 μM.

In another embodiment, the medium is new 3i medium including Neuro basal medium 50%, DMEM/F-12 50%, N2 supplement 1/200 v/v, B27 supplement 1/100 v/v, 100 mM L-glutamine about 1/50, 1/100, 1/200, 1/300, 1/400, or 1/500 v/v, 0.1 M β-ME about 1/500, 1/1000, 1/1500, or 1/2000 v/v, SU5402 (FGFR inhibitor) about 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, PD184352 (ERK cascade inhibitor) about 0.5 μM, 0.8 μM, 1 μM, 2 μM, PD0325901 (MAPK inhibitor) about 0.1 μM, 0.2, μM, 0.3 μM, 0.4 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, CHIR99021 (GSK3 inhibitor) about 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, and Dorsomorphin (BMP cascade inhibitor) 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM. In another embodiment, the medium is new 3i medium including Neuro basal medium about 30 to 60%, DMEM/F-12 about 30 to 60%, N2 supplement 1/100, 1/200, 1/300, 1/400, or 1/500 v/v, B27 supplement about 1/50, 1/100, 1/200, 1/300, 1/400, or 1/500 v/v, 100 mM L-glutamine 1/100 v/v, 0.1M β-ME 1/1000 v/v, SU5402 (FGFR inhibitor) 2 μM, PD184352 (ERK cascade inhibitor) 0.8 μM, PD0325901 (MAPK inhibitor) 0.4 μM, CHIR99021 (GSK3 inhibitor) 3 μM, and Dorsomorphin (BMP cascade inhibitor) 2 μM.

In another embodiment, the medium is 2i medium. In one embodiment, the 2i medium includes PD0325901 about 0.1 μM, 0.2, μM, 0.3 μM, 0.4 μM, 0.5 μM, and CHIR99021 about 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

In another embodiment, the medium is alternative 3i medium, including PD0325901 about 0.1 μM, 0.2, μM, 0.3 μM, 0.4 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, CHIR99021 about 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, and Dorsomorphin about 0.5 μM, 0.8 μM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

In one embodiment, the present invention provides a method of transplanting mature hepatocytes into liver damage models by producing mature hepatocytes and transplanting the mature hepatocytes into liver damage models. In another embodiment, the mature hepatocytes are produced by transferring stem cells to a medium supplemented with small molecule inhibitors of MEK kinase, GSK-3 and/or FGF signaling. In another embodiment, the stem cells are human ES cells.

The present invention is also directed to kits for the induction, propagation and/or isolation of liver progenitor cells. The present invention is also directed toward kits for the transplantation of liver progenitor cells to liver damaged models.

Each kit is an assemblage of materials or components. The exact nature of the components configured in each inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of inducing and/or propagating hepatocyte precursors.

Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit for a desired purpose, such as for induction, propagation and/or isolation of liver progenitor cells.

Optionally, the kits also contain other useful components, such as those described herein, or buffers (e.g., PBS), growth media, tissue culture plates, multiple-well plates, flasks, chamber slides, differentiation media, stem cell media, goat serum, fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, diluents, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, or other useful paraphernalia as will be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s).

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1 Inducing and Propagating Cells and Evidence of Adult Liver Stem Cells

The inventors previously identified a monoclonal antibody called GCTM-5. This reagent reacts with hepatoblasts, the precursors of bile ducts and hepatocytes, in the human embryonic liver. In the adult human liver, this antibody identifies a subset of bile duct cells in the termini of the biliary tree in the normal liver. In pathological states such as alcoholic cirrhosis, this population expands and can convert into cells co-expressing hepatocyte markers. The inventors also previously demonstrated that cells bearing the GCTM-5 antigen, and coreactive with liver specific markers such as amylase, albumin, and cytokeratin 19 could be induced in human ES cell cultures by specific modifications to the culture medium, including the use of low glucose basal medium containing low concentrations of serum supplemented with nicotinamide. The induction was slow and not efficient, the nature of these GCTM-5 positive cells was unknown, and they could not be serially cultivated. Here, the inventors describe important modifications to the procedure for inducing and propagating the cells and produce evidence that these cells are similar to adult liver stem cells.

Human ES cell cultures, maintained as described previously in serum containing medium in the presence of fibroblast feeder cell support, are transferred to defined medium supplemented with small molecule inhibitors of MEK kinase, GSK-3, and FGF signaling. This combination of inhibitors has been shown to maintain pluripotency in cultures of mouse ES cells but paradoxically, when added to cultures of human ES cells, it induces differentiation and the appearance of cells with morphology of primitive hepatocyte precursors. These cultures contain cells that are positive for the GCTM-5 antigen and may coexpress EpCAM and NCAM along with GCTM-5 on their surface. This combination of antigens enables the isolation of pure populations of progenitors from the differentiating cultures by flow cytometry or magnetic bead purification. These cells may also co-express markers of the bile duct lineage, such as cytokeratin 19, or markers of hepatocytes, such as amylase, albumin, or other liver specific gene products. The cells propagate and undergo expansion for prolonged periods in the presence of this medium described below, or in Kubota's medium, a minimal formulation previously shown to promote growth of adult liver progenitor cells in vitro. The cells are positive for surface markers characteristic of liver progenitor cells in the adult including the GCTM-5 antigen, Ep-CAM, and N-CAM. They are capable of differentiation into cells expressing markers of either bile duct or hepatocytes, such markers including albumin, transthyretin, cytochrome P450 enzymes. They show functional activity of adult liver cells such as indocyanine green dye uptake. They can give rise to mature hepatocytes either in vitro or following transplantation into liver damage models in vivo.

Example 2 Materials

-   -   HES2 or HES3 cells     -   MEF feeder seeded organ culture dishes     -   20% FCS hES medium     -   3i medium

Neuro basal medium 50% DMEM/F-12 50% N2 supplement 1/200 v/v B27 supplement 1/100 v/v 100 mM L-glutamine 1/100 v/v 0.1M β-ME 1/1000 v/v SU5402 (FGFR inhibitor) 2 μM PD184352 (ERK cascade inhibitor) 0.8 μM CHIR99021 (GSK3 inhibitor) 3 μM

-   -   Matrigel-coated organ culture dish (1 hour coating using 1 ml of         1/30 DMEM/F-12-diluted BD Matrigel hESC-qualified Matrix)

Example 3 Procedure

HES2 or 3 cells were cultured for 5 days on MEF-feeders with 20% FCS hES medium in organ culture dishes. 20% FCS hES medium was replaced with 3i medium and cells were keeping culture in this medium for 3 days. Cells were detached as clumps by collagenase and feeder cells were removed by sedimentation in DMEM/F-12 medium. Cells were seeded on Matrigel-coated organ culture dish as 1 to 1 split and culture with 3i medium. After 3 to 5 days in culture, hepatoblast-like cells appeared. They were subsequently propagated using 3i or Kubota's medium following enzymatic dissection.

Example 4 Gene Expression in Human Embryonic Stem Cells (hESC) Induced to Differentiate in 3-I Medium

The inventors used QRT-PCR to examine gene expression in hESC cultures induced to differentiate using 3-I containing medium. Cells were transferred from stock cultures maintained under standard ES cell growth conditions, then placed in 2-1 or 3-I medium. The cultures grown in the inducing medium containing cells of different morphology, with large cells resembling hepatoblasts. FIG. 1 shows that the large cells do not express pluripotency markers, but express markers of definitive endoderm (Sox-17), extraembryonic endoderm (Sox-7, Gata-4, AFP), and gut derivatives (Pax-6, pancreas; Cdx-2, intestine; albumin, liver; FoxA2 and HNF-6, liver progenitors). The striking induction of albumin, HNF-6, and HNF-1, compared to AFP, suggests the population may contain bipotent bile duct and hepatocyte progenitors (FIGS. 2 a and b). FIG. 3 shows immunostaining for HNF3 beta, Sox-17, and albumin. The 2-1 or 3-I cultures induce endodermal differentiation in the hESC cultures, and the cultures strongly express markers of liver progenitors. Further modification of the culture system, including suppression of BMP and insulin/IGF signaling early on, enhanced the yield of definitive endoderm and liver progenitors.

Example 5 Isolation, Culture and Characterization of GCTM-5 Positive Cells from the Nonparenchymal Fraction of Adult Human Liver

The inventors believed that the 3-I induced cultures might contain cells showing similar patterns of gene expression to the GCTM-5 positive cells in the adult liver. Study of the growth requirements of these adult liver cells might enhance our ability to propagate the 3-I induced liver progenitors directly. Therefore, the inventors further characterized GCTM-5 positive cells isolated from the non-parenchymal fraction of the adult human liver. In previous work, GCTM-5 cells isolated from adult liver were grown under conditions that supported only bile duct differentiation, and this differentiation was observed under those conditions. We plated freshly isolated adult non-parenchymal cells into a modified culture system that was designed to promote expansion of bipotent liver progenitor cells. Cells were isolated using magnetic bead immunoaffinity, then placed into culture in modified Kubota's medium on collagen type III or IV in the presence of FGF-2. Under these conditions, the cells attached and formed microcolonies which expanded for up to four weeks (FIG. 4). Matrigel failed to support robust attachment and colony formation of the GCTM-5 positive cells. Contrary to previous reports these cells could not be serially cultivated using the Kubota's medium. Because the cells could not be serially cultivated, the inventors examined expression of receptor associated kinase genes using a low density QRTPCR array, with a view towards discovering additional factors that might help expand the population in vitro.

Example 6 Kinase Array Analysis of GCTM-5 Positive Cells from the Non-Parenchymal Fraction of Adult Liver and 3-I Induced Human Embryonic Stem Cells

Unfractionated liver non-parenchymal cells were compared with GCTM-5 positive cells, GCTM-5 negative cells (essentially the same as the unfractionated population), and 3-I cells for their expression of kinases. Kinases that were expressed in all fractions and enriched in the GCTM-5 positive fraction included the following: ACVR1B, BMP2R, EGFR, ERBB2, ERBB3, FGFR1, FGFR2, FGFR3, IGFR1, INSR, KDR, TEK, TGFBR1, TGFBR2. Because the 3-I cells in this experiment were not fractionated, it was not expected that their patterns of gene expression would be similar to those of adult liver GCTM-5 positive cells. The results suggested that Activin, BMP, EGF, heregulin, FGF-1, IGF, VEGF, angiopoietin, and TGF beta might stimulate growth or differentiation of the GCTM-5 positive cells.

Example 7 Methods: 3i-Induction of Hepatocyte-Like Cells—Materials

HES2 or HES3 cells were cultured on organ culture dishes seeded with mouse embryo fibroblast feeder cells in human embryonic stem cell medium containing 20% fetal calf serum.

3i* or new 3i** medium

-   -   Neuro basal medium 50%     -   DMEM/F-12 50%     -   N2 supplement 1/200 v/v     -   B27 supplement 1/100 v/v     -   100 mM L-glutamine 1/100 v/v     -   0.1M β-ME 1/1000 v/v     -   *SU5402 (FGFR inhibitor) 2 μM     -   *PD184352 (ERK cascade inhibitor) 0.8 μM     -   **PD0325901 (MAPK inhibitor) 0.4 μM     -   CHIR99021 (GSK3 inhibitor) 3 μM     -   **Dorsomorphin (BMP cascade inhibitor) 2 μM

Matrigel-coated organ culture dish (Coated for one hours with 1 ml of 1/30 DMEM/F-12-diluted BD Matrigel hESC-qualified Matrix)

Example 8 Methods: 3i-Induction of Hepatocyte-Like Cells—Procedure

HES2 or 3 cells were cultured 5 days on MEF-feeders with 20% FCS hES medium in organ culture dishes. 20% FCS hES medium was replaced with 3i medium and cells were maintained in this medium for 2 days.

Cells were detached as clumps by collagenase and feeder cells were removed by sedimentation in DMEM/F-12 medium.

Cells were seeded on Matrigel-coated organ culture dish as 1 to 1 split and culture with 3i medium.

After 3 to 5 day culture, the hepatocyte-like cells appeared.

Example 9 Methods: qPCR Analysis

The induced hepatocyte-like cells were marked and picked manually into microtubes. Undifferentiated HES3 cells and human dermal fibroblast cells were collected as negative controls, and HepG2 cells were collected as a positive control cell. The Cells were lysed and total RNA was purified by RNA Easy Micro kit (Qiagen) with on column Dnase-treatment. Then cDNA was synthesized from 1 μg total RNA by random hexamer priming using an OmniScript kit (Quiagen). The cDNA was diluted 8 fold into water and 2× TaqMan PCR master mix and TaqMan Gen Expression Assay Mix were added (Applied Biosystems). PCR were performed on an ABI prism 7900 instrument (Applied Biosystems). The data was analyzed with delta CT method by RQ manager software (Applied Biosystems). All qPCR reaction were duplicated in each experiment and analysis were performed analyzed with triplicate experiments.

TaqMan Gene Expression Assays:

-   -   Gene AssayID Expression     -   Cyclophilin (PPIA) Hs99999904_m1 Ubiquitous (Endogeneous         control)     -   OCT3/4 (POU5F1) Hs01895061_u1 Undifferentiated cells     -   GATA4 Hs00171403_m1 Early definitive/extraembryonic Endoderm     -   T (Brachyury) Hs00610080_m1 Early mesoendoderm/mesoderm     -   SOX7 Hs00846731_s1 Early extraembryonic Endoderm     -   SOX17 Hs00751752_s1 Early definitive Endoderm     -   CDX2 Hs00230919_m1 Early trophectoderm/gut endoderm     -   PAX6 Hs00240871_m1 Neuroectoderm/early pancreatic cells     -   FOXA2 (HNF3) Hs00232764_m1 Early liver cells     -   HNF6 (ONECUT1) Hs00413554_m1 Early liver cells     -   AFP Hs01040597_m1 Early liver cells/extraembryonic Endoderm     -   ALB (Albumin) Hs00609403_m1 Hepatoblast/Placenta

Example 10 Methods: GCTM-5 Positive Cells Isolation

Human liver non-parenchymal cells, supplied by CellzDirect/Life Technologies. Were collected into 50 ml tubes (10E8 cells/tube). The cells were suspended with buffer 1 (0.1% BSA/2 mM EDTA/PBS(−)), and centrifuged 1000 rpm 5 minutes, and this washing step was repeated until the supernatant were clear (around 3 times). GCTM-5 antibody or control IgG1 were added as 1 μg/10E6 cells (100 μg/50 ml buffer 1/tube), and incubated with gentle rotation at 4° C. for 1 hour. The cells were collected by centrifugation and non-reacted antibody was washed out by 3 times with buffer 1 and centrifugation. Then cells were suspended with 10 ml buffer 1/tube and magnetic beads-conjugated secondly antibodies (Dynabeads Rat anti-Mouse IgG1: Dynal Biotech) were added as 10 μl/10 E7 cells (100 μl/10 ml buffer 1/tube), and then incubated with gentle rotation at 4° C. 1 hour. The cell suspension was transferred into 1.5 ml microtubes. The tubes were placed in the magnet stand, the supernatant was discarded, and the cell pellets in the tube walls were resuspended with buffer 1. The washing step with buffer 1 were repeated 3 times and then washed with culture medium once. The cell pellets were resuspended with culture medium and seeded onto culture plates, or directly lysed with Trysol (Invitorogen) for RNA pulification.

Example 11 Methods: Taqman Human Protein Kinase Array Analysis

The GCTM-5 positive, negative, non-selected cell total RNA were purified as a magnet bead-free pellet in Trysol (Invitrogen). The pre-purified total RNA was re-purified by RNA easy micro kit with on column Dnase treatment (Qiagen). The 3i-induced hapatocyte-like cell RNA was purified as described above. Then cDNA was synthesized from 2 μg total RNA by OmniScript kit (Quiagen) with random hexamer primers. The cDNA was diluted 10 times by water, 2× TaqMan PCR master mix was, and samples were then loaded onto TaqMan Human Protein Kinase Array (4367784: Applied Biosystems). PCR reactions were performed on the ABI prism 7900 (Applied Biosystems). The data was analyzed with the delta delta CT method by RQ manager software (Applied Biosystems). The experiments were duplicated to confirm the results.

Example 12 Various FGF/MEK/ERK, GSK3b and BMP Signaling Inhibitors May be Utilized for the Induction and Selection of Endoderm Cells

Induction and Selection of Endoderm Cells Via:

-   -   FGF/MEK/ERK inhibition—inhibit ES cell self-renewal and induce         differentiation, inhibit neural and trophectodermal cell growth.     -   GSK3b inhibition—induce mesoderm/endoderm differentiation.     -   Serum starvation—inhibit mesoderm/mesenchymal cell growth.     -   BMP inhibition (optionally)—inhibit extraembryonic cell         differentiation.

The FGF/MEK/ERK signaling pathway includes several molecules, such as FGF, FGFR, FRS, GRB, SOS, RAS, RAF, MEK, ERK, etc. All of these molecules may be utilized as potential targets to inhibit this pathway. Additionally, GSK3b inhibition means that the induction of the Wnt signaling pathway could also be utilized for the same purpose. This, in turn, would include Wnt signaling inhibitory molecules, such as GSK3b, Axin, APC, which could also be potential targets.

Numerous examples of FGFR/MEK/ERK signaling inhibitors and GSK3b inhibitors exist, both commercially and noncommerically. Some examples include the following:

-   -   FGFR inhibitors: PD173074, PLX052, PKC412, AZD2171, Ki23057,         BIBF1120, CHIR-258, Mastinib (AB1010), PHA739358, BMS-582664,         AZD2171, PRO-001, Tyrphostin A46, Tyrphostin B40.     -   MAPK/MEK(MAPKK)/ERK pathway inhibitors: PD98059, U0126,         5-Indotubercidin,         terna Zentaris, PD98059, AG 99, Apigenin, SP600125,         3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione,         SL327, SU4984, FR180204, SB203580, PD169316, SB202190, ERK         activation inhibitor peptides (such as Ste-PKKKPTPIQLNP-NH₂).     -   GSK3b inhibitors: BIO and related products, CT99021, Aloisine,         RP106, Aloisine A, TDZD-8, OTDZT, AR-A014418, 7AIPM,         Neurogenesis Inducer, TWS119, Kenpaullone (NSC-664704),         Indirubin-3′-monoxime, GSK3b inhibitor peptide (such as         Myr-N-GKEAPPAPPQSpP-NH₂), Wnt signaling ligands (such as Wnt1         and/or Wnt3a).     -   BMP inhibitors: LDN-193189, BMP inhibitor proteins (such as         Noggin, Chordin, Follistatin, and/or Wif).

Example 13 3i, Alternative 3i and 2i Medium Components—Dosages Examined

(1) In combination of SU5402, PD184352 and CHIR99021:

-   -   SU5402: 1 μM decreased hepatic differentiation and increased         trophectodermal/neural cells; 2 μM most efficient; 4 μM toxic.     -   PD 184352: 0.4 μM decreased hepatic differentiation and         increased trophectodermal cells; 0.8 μM most efficient, 1.6 μM         no difference from 0.8 μM     -   CHIR99021: 1.5 μM dramatically decreased hepatic         differentiation/growth; 3 μM most efficient; 6 μM some kind of         non-hepatic cells grow very well.

(2) In combination of PD0325901 and CHIR99021:

-   -   PD0325901; 0.4 μM no difference from 3i, 0.8 μM more efficient         than 3i, 1.6 μM no difference from 0.8 μM

(3) In combination of PD0325901, CHIR99021 and Dorsomorphin:

-   -   Dorsomorphin: 1 μM slightly inhibit extraembryonic cell         differentiation without disturbing hepatic differentiation; 2 μM         greatly inhibit extraembryonic cell differentiation without         disturbing hepatic differentiation; 5 μM toxic.

The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the methods of inducing and/or propagating a quantity of endoderm progenitor cells, methods of culturing, components of culture mediums, including pharmaceutical equivalent, analog, derivative and/or salt thereof, and types of diseases, conditions and/or target organ(s) that relate to the teachings of the invention, techniques and composition and use of solutions used therein, and the particular use of the products created through the teachings of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventor for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described. 

1. A method of inducing and/or propagating a quantity of endoderm progenitor cells, comprising: a) providing a quantity of human pluripotent stem cells; b) placing the quantity of human pluripotent stem cells in a medium supplemented with an inhibitor of MKK1/2, a fibroblast growth factor receptor (FGFR) inhibitor, and a glycogen synthase kinase 3 (GSK3) inhibitor; and c) culturing the human pluripotent stem cells to form endoderm progenitor cells.
 2. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.
 3. The method of claim 1, wherein the inhibitor of MKK1/2 comprises a compound of the formula:

or a salt thereof.
 4. The method of claim 1, wherein the inhibitor of MKK1/2 comprises a compound of the formula:

or a salt thereof.
 5. The method of claim 1, wherein the FGFR inhibitor comprises a compound of the formula:

or a salt thereof.
 6. The method of claim 1, wherein the GSK3 inhibitor comprises a compound of the formula:

or a salt thereof.
 7. The method of claim 1, wherein the endoderm progenitor cells are hepatocyte-like.
 8. The method of claim 1, wherein the human pluripotent stem cells comprises human embryonic stem cell 2 (HES2) and/or human embryonic stem cell 3 (HES3).
 9. The method of claim 1, wherein the medium comprises 0.2 to 3.0 μM of the inhibitor of MKK1/2.
 10. The method of claim 1, wherein the medium comprises 1.0 to 6.0 μM of the FGFR inhibitor.
 11. The method of claim 1, wherein the medium comprises 1.0 to 6.0 μM of the GSK3 inhibitor.
 12. The method of claim 1, wherein the medium further comprises a bone morphogenetic protein (BMP) inhibitor.
 13. The method of claim 12, wherein the medium comprises 1.0 to 6.0 μM of the BMP inhibitor.
 14. The method of claim 12, wherein the BMP inhibitor comprises dorsomorphin.
 15. A composition comprising an inhibitor of MKK1/2, an FGFR inhibitor, a GSK3 inhibitor, and a BMP inhibitor.
 16. A composition comprising activin, BMP, epidermal growth factor (EGF), heregulin, fibroblast growth factor 1 (FGF-1), insulin growth factor (IGF), vascular endothelial growth factor (VEGF), angiopoietin, and transforming growth factor beta (TGF beta).
 17. A composition, comprising a quantity of endoderm progenitor cells, produced by the process of claim
 1. 